EP1210095B1 - Withania somnifera composition - Google Patents

Withania somnifera composition Download PDF

Info

Publication number
EP1210095B1
EP1210095B1 EP00982710A EP00982710A EP1210095B1 EP 1210095 B1 EP1210095 B1 EP 1210095B1 EP 00982710 A EP00982710 A EP 00982710A EP 00982710 A EP00982710 A EP 00982710A EP 1210095 B1 EP1210095 B1 EP 1210095B1
Authority
EP
European Patent Office
Prior art keywords
extract
composition
withania somnifera
oligosaccharides
aqueous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP00982710A
Other languages
German (de)
French (fr)
Other versions
EP1210095A1 (en
EP1210095A4 (en
Inventor
Shibnath Ghosal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Indian Herbs Research and Supply Co Ltd
Natreon Inc
Original Assignee
Indian Herbs Research and Supply Co Ltd
Natreon Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Indian Herbs Research and Supply Co Ltd, Natreon Inc filed Critical Indian Herbs Research and Supply Co Ltd
Publication of EP1210095A1 publication Critical patent/EP1210095A1/en
Publication of EP1210095A4 publication Critical patent/EP1210095A4/en
Application granted granted Critical
Publication of EP1210095B1 publication Critical patent/EP1210095B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed

Definitions

  • This invention relates to a composition of the plant Withania Somnifera, a process for making such a composition and use of such a composition. More particularly, the invention relates to a high purity extract composition with advantageous levels of withanolides glycosides, sitoindosides and oligosaccharides, and substantially low levels of free withaferin A, which provides enhanced cognition-enhancing effects for the user, and an extraction process for obtaining such composition, as well as pharmaceutical and nutritional use products thereof.
  • Ashwagandha The plant Withania Somnifera Dunn. (Solanaceae), commonly known as Ashwagandha, has been used in herbal formulations of the Ayurvedic or Indian system of medicine to attenuate a cerebral function deficit in the geriatric population, and to augment the faculty of learning and memory to provide a nonspecific host defense. These beneficial effects help the organism to ward off stress and act as an adaptogen. Ashwagandha also shows significant protection against pentylene tetrazole - induced seizures in experimental models of epilepsy, indicating its potential utility for treatment of petitmal epilepsy. Ashwagandha administration also produces a decrease in the core body temperature suggesting a reduced Body Metabolic Rate (BMR), enhanced body growth and increased longevity.
  • BMR Body Metabolic Rate
  • the invention provides a Withania Somnifera extract composition obtainable by extraction with a 1:1 aqueous-alcoholic solvent, said extract being in the form of a stable, herbaceous powder, which produces a cognition effect and learning facility for the user, when taken in a dosage of 200-800 mg/day, which comprises, by weight, (a) at least 3% of withanolide glycosides including sitoindosides, (b) at least 3% of oligosaccharides, and (c) less than 0.5% of withaferin A (aglycone), wherein the weight ratio of (a):(c) is 75-95:25-5, and the weight ratio of (a):(b) is 40-60:60-40.
  • the invention also provides a pharmaceutical or nutritional use product which includes the above extract composition in a dosage form suitable for oral administration at a dosage level is 200 to 800 mg/day, optionally including, excipients suitable for such oral administration, wherein the dosage form is a tablet, a capsule or an elixir or suspension.
  • the product may be a vitamin tablet.
  • the composition may be used in a process for making a pharmaceutical product for immuno-stimulation.
  • the above high purity Withania Somnifera plant extract composition with substantially low levels of cytotoxic withaferin A (aglycone), in the form of a stable, free-flowing light yellow-to-brown herbaceous powder composition provides enhanced cognition and augmented learning facility in the geriatric population when taken in a dosage of about 200-800 mg/day.
  • the biologically-enhancing composition of the invention preferably includes, by weight, (a) 3 to 8% of withanolide glycosides and sitoindosides, (b) 3 to 8%, of oligosaccharides, preferably a mol. wt. of ⁇ 2000.
  • the composition is typically at least 90% soluble, the ash content of this composition is typically less than 8%, and its moisture content is typically less than 5% (w/w).
  • the extract composition is obtained by (a) providing root stock of a Withania Somnifera plant which is about 1 to 2 years old*, (b) extracting the root stock immediately with an aqueous-alcoholic solvent, (c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove withanolide aglycones therefrom, (e) vacuum drying the insoluble residue below about 60°C to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions.
  • older roots contain undesired high molecular weight polymeric saccharides
  • the aqueous-alcoholic solvent is water-methanol or water-ethanol, preferably in a 1:1 ratio, and the organic solvent is chloroform or ethyl acetate.
  • the chloroform soluble-residual which contains mainly cytotoxic withanolide aglycones and other constituents of the plant which do not contribute to the bioactivity of the Ashwagandha composition, is discarded.
  • the chloroform-insoluble/aqueous-soluble residue contains the desired withanolide glycoside and sitoindoside components which are potent bioactive constituents of Ashwagandha.
  • the amount of such withasteroid glycosides therein, i.e. withanolide glycosides and sitoindosides, is determined on the basis of the withasteroid aglycones produced by subsequent hydrolysis of the chloroform-insoluble but aqueous soluble extract-fraction.
  • the resultant extract powder also contains desirable levels of oligosaccharides having a molecular weight of ⁇ 2000.
  • the water-soluble portion of this extract (see Table 1, column 2) contains 20 to 35% oligosaccharides, whereas commercially available extracts from other plants contain an excessive amount, i.e. 90%, of high molecular weight polysaccharides with only traces of oligosaccharides.
  • Withanolide glycosides/sitoindosides the major bioactive constituents of Withania Somnifera, are not readily identifiable in HPTLC chromatographs. However, upon carefully controlled hydrolysis, wherein they are converted into withanolide aglycones, they are readily observed in their HPTLC fingerprints. On the basis of such post-hydrolysis findings the presence and amounts of such withanolides/ sitoindoside glycosides in the extract composition of the invention was determined. In contrast, commercially available Withania Somnifera extracts lack these withanolide glycoside/sitoindoside component.
  • the withanolide aglycones are highly susceptible to rearrangement under acidic conditions.
  • Withania Somnifera Extract Composition of Invention Appearance: Fine, free flowing, hygroscopic powder Color: Light yellow to brown Water soluble extractive value: Min 90.00 % w/w Total Withanolide glycosides and conjugates: Min 3% w/w (estimated as acetates), preferably 3-8% Oligosaccharides (molecular weight ⁇ 2000): Min 3% w/w (estimated as acetates), preferably 3-8% Free Withaferin A: Max 0.5 % w/w Ratio of Withanolide [glycosides and conjugates] to free withaferin A (aglycone): 75-95:25-5 Ratio of Withanolides To Oligosaccharides: 40-60:60-40 Total ash content: Max 8.00 % w/w Moisture content: Max 5.00 % v/w Dosage:
  • Chewable Tablets Ingredient Composition (w/w, in %) Quantity per Tablet (mg) 1. Withania Somnifera extract 12.26 27.60 2. Sodium ascorbate, USP 36.26 81.60 3. Avicel pH 101 17.12 38.50 4. Sodium saccharin, (powder), N.F. 0.56 1.25 5. DiPac 29.30 66.00 6. Stearic acid, N.F. 2.50 5.60 7. Imitation orange Flavor 1.0 2.25 8. FD & C Yellow #6 dye 0.5 1.12 9. Cab-O-Sil 0.5 1.12
  • Elixir Formula Ingredient Quantity 1. Withania Somnifera Extract 0.2 g 2. Lemon Tincture 5.0 ml 3. Orange Tincture 5.0 ml 4. Sodium Saccharin 0.5 g 5. Propylene Glycol 65.0 ml 6. Glycerine 15.0 ml 7. Sorbitol, USP, sufficient quantity to make 100.0 ml
  • Elixir Formula Ingredient Quantity 1. Withania Somnifera Extract 0.4 g 2. Orange oil 0.1 ml 3. Benzaldehyde 0.005 ml 4. Sorbitol Solution USP 10.0 ml 5. Propylene Glycol 40.0 ml 6. Alcohol 40.0 ml 7. Purified Water, sufficient quantity to make 100.0 ml
  • Colloidal Magnesium Aluminum Premix (5% fully hydrated magma) Ingredient Quantity 1. Colloidal magnesium aluminum silicate 5.0% w/v 2. Methyl paraben 0.12% w/v 3. Propyl paraben 0.03% w/v 4. Water, qs 100%
  • step 2 Add liquid sugar colloidal magnesium aluminum silicate premix and half the poloxamer 331 and sulfa drug in step 2 with agitation. 3. Disperse the rest of the poloxamer 331 and sulfa drug in step 2 with agitation. 4. 4. Add flavor to step 3 and pass the suspension through a colloid mill or homogenizer rinsing through with purified water. 5. Adjust pH to 5.5 with either citric acid or sodium hydroxide solution. 6. Add purified water to final volume.
  • Ehrlich Ascites tumor (s-180, 1 x 10 6 cells, all viable), suspended in phosphate buffer saline (PBS, 0.5 ml), were inoculated (i.p.) to a group of adult mice. After 2 h, withaferin-A (WA) (2.5 mg/100 g b.w.) and withanolide + oligosaccharides (1:10 w/w) were administered to Group-2 and Group-3 animals (Table 3). The control (Group 1) received only the vehicle (PBS) following the tumor inoculation. On day-10, the tumor cells were removed from the peritoneal cavity and the viable and dead cells were enumerated microscopically using the dye-exclusion method.
  • PBS phosphate buffer saline
  • the oligosaccharide carrier Group 3 in the extract composition of the invention can significantly modify the nature and extent of the bioactivity of withaferin-A (WA).
  • the Withania Somnifera extract composition of the invention includes an advantageous combination of components in defined amounts and proportions for optimum biological activity.
  • the sitoindoside constituent therein produces an immunostimulation response as reflected by activation of peritoneal macrophages, phagocytosis, and increased activity of lysomal enzymes secreted by activated macrophages.
  • the substantial absence of withasteroid aglycones therein precludes the adverse cytotoxic effect observed with other related compositions in the art.
  • the defined amount and kind of oligosaccharides in the extract composition of the invention also plays a very important role in the bioavailability of the Withania Sominifera active principles. Specifically, the combination of such oligosaccharides without Withanolide aglycones elicits a desirable immunostimulatory effect for the user.

Abstract

A high purity Withania somnifera extract composition in the form of a high purity, stable, free-flowing, light yellow-to-brown herbaceous powder, for producing an enhanced cognition effect for the user and to augment the learning facility in the geriatric population when taken in a dosage of 200-800 mg/day. This extract contains, by weight, (a) at least 3% of withanolide glycosides and sitoindosides, preferably 3-8%, (b) at least 3% of oligosaccharides, preferably 3-8%, preferably a molecular weight of <2000, and (c) less than 0.5% of free cytotoxic withaferin A (aglycone), wherein the ratio of (a):(c) is 75-95:25-5, and the ratio of (a):(b) is 40-60:60-40. An extraction process for obtaining such extract composition, and pharmaceutical and nutritional use products thereof, also are described.

Description

  • This invention relates to a composition of the plant Withania Somnifera, a process for making such a composition and use of such a composition. More particularly, the invention relates to a high purity extract composition with advantageous levels of withanolides glycosides, sitoindosides and oligosaccharides, and substantially low levels of free withaferin A, which provides enhanced cognition-enhancing effects for the user, and an extraction process for obtaining such composition, as well as pharmaceutical and nutritional use products thereof.
  • The plant Withania Somnifera Dunn. (Solanaceae), commonly known as Ashwagandha, has been used in herbal formulations of the Ayurvedic or Indian system of medicine to attenuate a cerebral function deficit in the geriatric population, and to augment the faculty of learning and memory to provide a nonspecific host defense. These beneficial effects help the organism to ward off stress and act as an adaptogen. Ashwagandha also shows significant protection against pentylene tetrazole - induced seizures in experimental models of epilepsy, indicating its potential utility for treatment of petitmal epilepsy. Ashwagandha administration also produces a decrease in the core body temperature suggesting a reduced Body Metabolic Rate (BMR), enhanced body growth and increased longevity.
  • Typically, commercially available extracts of Ashwagandha obtained from old roots stock are either completely devoid of sitoindosides, or contain only traces of sitoindosides admixed with large amounts of toxic metabolites of withanolide aglycones, and polysaccharides, and wherein the polyoxygenated withasteroids are degraded during conventional extract procedures. Moreover, admixture of several undetermined chemo-types of such wild-crafted Withania roots create further complications in respect of their chemical ingredients.
  • The invention provides a Withania Somnifera extract composition obtainable by extraction with a 1:1 aqueous-alcoholic solvent, said extract being in the form of a stable, herbaceous powder, which produces a cognition effect and learning facility for the user, when taken in a dosage of 200-800 mg/day, which comprises, by weight, (a) at least 3% of withanolide glycosides including sitoindosides, (b) at least 3% of oligosaccharides, and (c) less than 0.5% of withaferin A (aglycone), wherein the weight ratio of (a):(c) is 75-95:25-5, and the weight ratio of (a):(b) is 40-60:60-40.
  • The invention also provides a pharmaceutical or nutritional use product which includes the above extract composition in a dosage form suitable for oral administration at a dosage level is 200 to 800 mg/day, optionally including, excipients suitable for such oral administration, wherein the dosage form is a tablet, a capsule or an elixir or suspension.
  • The product may be a vitamin tablet.
  • The composition may be used in a process for making a pharmaceutical product for immuno-stimulation.
  • The above high purity Withania Somnifera plant extract composition with substantially low levels of cytotoxic withaferin A (aglycone), in the form of a stable, free-flowing light yellow-to-brown herbaceous powder composition, provides enhanced cognition and augmented learning facility in the geriatric population when taken in a dosage of about 200-800 mg/day. The biologically-enhancing composition of the invention preferably includes, by weight, (a) 3 to 8% of withanolide glycosides and sitoindosides, (b) 3 to 8%, of oligosaccharides, preferably a mol. wt. of <2000. The composition is typically at least 90% soluble, the ash content of this composition is typically less than 8%, and its moisture content is typically less than 5% (w/w).
  • Suitably, the extract composition is obtained by (a) providing root stock of a Withania Somnifera plant which is about 1 to 2 years old*, (b) extracting the root stock immediately with an aqueous-alcoholic solvent, (c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove withanolide aglycones therefrom, (e) vacuum drying the insoluble residue below about 60°C to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions.
    * older roots contain undesired high molecular weight polymeric saccharides
  • In a specific embodiment of the invention, the aqueous-alcoholic solvent is water-methanol or water-ethanol, preferably in a 1:1 ratio, and the organic solvent is chloroform or ethyl acetate.
  • In accordance with the process of the invention, freshly harvested thin roots of Withania Somnifera (Ashwagandha) obtained from steep rocks in the Himalaya mountains of India which is about 1-2 years old is coarsely crushed and then exhaustively extracted with hydroalcohol solvent e.g. a 1:1 mixture, at about 50-60°C. The aqueous-alcoholic extract then is concentrated under vacuum, the concentrated extract is further treated with chloroform. The chloroform-insoluble residue therefrom is then vacuum dried below 60°C to provide a dry extract, which is pulverized under controlled temperature and humidity conditions to form a fine powder.
  • The chloroform soluble-residual, which contains mainly cytotoxic withanolide aglycones and other constituents of the plant which do not contribute to the bioactivity of the Ashwagandha composition, is discarded.
  • The chloroform-insoluble/aqueous-soluble residue contains the desired withanolide glycoside and sitoindoside components which are potent bioactive constituents of Ashwagandha. The amount of such withasteroid glycosides therein, i.e. withanolide glycosides and sitoindosides, is determined on the basis of the withasteroid aglycones produced by subsequent hydrolysis of the chloroform-insoluble but aqueous soluble extract-fraction.
  • The resultant extract powder also contains desirable levels of oligosaccharides having a molecular weight of <2000. Specifically, the water-soluble portion of this extract (see Table 1, column 2) contains 20 to 35% oligosaccharides, whereas commercially available extracts from other plants contain an excessive amount, i.e. 90%, of high molecular weight polysaccharides with only traces of oligosaccharides.
  • The compositions of this invention, and commercially available extracts of Withania Somnifera, are summarized and compared in Table 1 below. TABLE 1
    Example/ Sample Description Glycowithanolides (=Sitoindosides) and Polysaccharides1 Sitoindosides2 Withanolide Aglycone3 % Ratio of Sitoindosides and Aglycones
    #1/IH-3581* 15.19 8.16 2.04 80:20
    #2/IH-12762* 10.28 4.37 0.82 84:16
    #4/IH-21744* 12.12 6.06 1.32 81:19
    #5/IH-22615* 19.40 5.41 1.20 82:18
    #6/IExtract A+ 22.32 0.10 0.08 10:90
    #7/IExtract B+ 8.44 0.08 1.22 06:94
    #8/IExtract C+ 5.33 0.04 2.02 02:98
    #9/IExtract D+ 11.77 0.17 1.88 06:94
    #10/IExtract E+ 14.33 0.002 0.59 04:96
    * Extracts of present invention
    + Commercially available extracts
    1 Water-soluble residue (%, w/w) containing oligosaccharides and polysaccharides*.
    * The compounds responded to both benzidine metaperiodate (for polyols) and Liebermann-Burchardt (for phytosteroids) reagents.
    2 Water-soluble, chloroform-insoluble residue (%, w/w in respect of root powder**).
    ** Sitoindosides were obtained by column chromatography to separate polysaccharides; characterization by high pressure thin layer chromatography (HPTLC) followed by acetylation and comprehensive spectroscopy.
    3 Chloroform-soluble residue***.
    *** Analyzed by HPTLC, preparative TLC, and comprehensive spectroscopy (proton nuclear magnetic resonance, mass spectroscopy, gas chromatograph-mass spectroscopy of silyl derivatives) using reference samples.
    • HPTLC Analysis of Withania Somnifera Extracts
  • Withanolide glycosides/sitoindosides, the major bioactive constituents of Withania Somnifera, are not readily identifiable in HPTLC chromatographs. However, upon carefully controlled hydrolysis, wherein they are converted into withanolide aglycones, they are readily observed in their HPTLC fingerprints. On the basis of such post-hydrolysis findings the presence and amounts of such withanolides/ sitoindoside glycosides in the extract composition of the invention was determined. In contrast, commercially available Withania Somnifera extracts lack these withanolide glycoside/sitoindoside component. Analytical and Chromatographic Conditions:
    Plate material: Silica gel 60F254
    Solvent: n-Butanol/Acetic acid/Water 4/1/2
    (before hydrolysis)
    Chloroform/Methanol 90/10
    (after hydrolysis)
    Application mode: Camag linomat IV
    Development mode: Twin Trough chamber
    Detection wavelength: 260 nm
  • The withanolide aglycones are highly susceptible to rearrangement under acidic conditions.
  • The unique characteristics of the Withania Somnifera extract composition of the invention are summarized in Table 2 below. TABLE 2
    Withania Somnifera Extract Composition of Invention
    Appearance: Fine, free flowing, hygroscopic powder
    Color: Light yellow to brown
    Water soluble extractive value: Min 90.00 % w/w
    Total Withanolide glycosides and conjugates: Min 3% w/w (estimated as acetates), preferably 3-8%
    Oligosaccharides (molecular weight <2000): Min 3% w/w (estimated as acetates), preferably 3-8%
    Free Withaferin A: Max 0.5 % w/w
    Ratio of Withanolide [glycosides and conjugates] to free withaferin A (aglycone): 75-95:25-5
    Ratio of Withanolides To Oligosaccharides: 40-60:60-40
    Total ash content: Max 8.00 % w/w
    Moisture content: Max 5.00 % v/w
    Dosage: 200-800 mg per day
    • Pharmaceutical and Nutritional Formulas
  • Representative pharmaceutical and nutritional use products which include the Withania Somnifera extract composition of the invention are given in Examples 11-19 below.
    • EXAMPLE 11
  • Tablets and Capsules
    Ingredient Composition (w/w, in %) Quantity per Tablet (mg)
    1. Withania Somnifera extract 60.0 250.0
    2. Avicel pH 101 20.0 84.0
    3. Starch 1500 17.5 75.5
    4. Stearic acid, 2.0 8.5
    N.F. (powder)
    5. Cab-O-Sil 0.5 2.0
    Withania Somnifera extract is granulated with starch paste to make a free-flowing powder. Blend all the ingredients, except 4, for 25 min. in a blender. Screen in 4 and blend for an additional 5 min. Compress into tablets using 7/16-in standard concave tooling. Alternately, the blended material can be filled into appropriate capsules.
    • EXAMPLE 12
  • Chewable Tablets
    Ingredient Composition (w/w, in %) Quantity per Tablet (mg)
    1. Withania Somnifera extract 12.26 27.60
    2. Sodium ascorbate, USP 36.26 81.60
    3. Avicel pH 101 17.12 38.50
    4. Sodium saccharin, (powder), N.F. 0.56 1.25
    5. DiPac 29.30 66.00
    6. Stearic acid, N.F. 2.50 5.60
    7. Imitation orange Flavor 1.0 2.25
    8. FD & C Yellow #6 dye 0.5 1.12
    9. Cab-O-Sil 0.5 1.12
  • Blend all the ingredients, except 6, for 20 min in a blender. Screen in 6 and blend for an additional 5 min. Compress into tablets using 7/16-in standard concave tooling.
    • EXAMPLE 13
  • "Maintenance" Multivitamin Tablets or Capsules
    Ingredient Composition (w/w, in %) Quantity per Tablet (mg)
    1. Withania Somnifera extract, free-flowing 33.25 66.50
    2. Vitamin A acetate (dry form 500 IU and 500 D2 per mg) 5.5 11.0
    3. Thiamine mono-nitrate, USP 0.8 1.65
    4. Riboflavin, USP 1.1 2.10
    5. Pyridoxine HCl, USP 1.0 2.10
    6. 1 % Cyanocobalamine (in gelatin)
    7. D-Calcium pantothenate, USP 3.75 7.50
    8. Niacinamide 11.0 22.00
    9. DiTab 13.1 26.20
    10. Microcrystalline cellulose, N.F. 25.0 50.00
    11. Talc, USP 3.0 6.00
    12. Stearic acid, (powder), N.F. 1.5 3.00
    13. Magnesium stearate, (powder), N.F. 1.0 2.00
  • Blend all ingredients for 20 min in a suitable blender. Screen in 12 and blend for an additional 5 min. Compress at a tablet weight of 200 mg using 3/8-in standard concave tooling. Alternately, blended material is filled into a capsule containing 200 mg of multi-vitamins. These tablets or capsules can be used as nutritional supplements.
    • EXAMPLE 14
  • Geriatric Formula Vitamin Tablets
    Ingredient Composition (w/w, in %) Quantity per Tablet (mq)
    1. Withania Somnifera extract free-flowing powder 17.45 96.00
    2. Ferrous sulfate, USP 95% Ethecal granulation 15.00 78.00
    3. Thiamine mono-nitrate, USP 1.09 6.00
    4. Riboflavin, USP 1.00 5.50
    5. Niacinamide, USP 6.00 33.00
    6. Ascorbic acid USP fine crystal 15.00 78.00
    7. Calcium pantothenate, USP 0.73 4.00
    8. Pyridoxine HCl, USP 0.14 0.75
    9. Cyanocobalmine, 0.1% spray dried 0.82 4.50
    10. AcDisol 2.00 11.00
    11. Stearic acid, (powder),N.F. 2.00 11.00
    12. Magnesium stearate, (powder), N.F. 0.25 1.38
    13. CeloCat 38.52 211.87
  • Prepare a premix of items 2, 3, 6, 7. Mix in other ingredients except 10 and 11 and blend for an additional 5 min. Compress using oval punches (1 = 0.480 in., w = 0.220 x cup = 0.040 in.) Sugar or film coat. These tablets can be used as nutritional supplements.
    • EXAMPLE 15
  • Elixir Formula
    Ingredient Quantity
    1. Withania Somnifera Extract 0.2 g
    2. Lemon Tincture 5.0 ml
    3. Orange Tincture 5.0 ml
    4. Sodium Saccharin 0.5 g
    5. Propylene Glycol 65.0 ml
    6. Glycerine 15.0 ml
    7. Sorbitol, USP, sufficient quantity to make 100.0 ml
  • Dissolve 1 in 5 and 6 which have been heated to 50°C. Dissolve 4 in 2 and 3 and add the solution of Withania Somnifera extract at 25°C. Add sufficient sorbitol to make the product measure 100 ml.
    • EXAMPLE 16
  • Elixir Formula
    Ingredient Quantity
    1. Withania Somnifera Extract 0.4 g
    2. Orange oil 0.1 ml
    3. Benzaldehyde 0.005 ml
    4. Sorbitol Solution USP 10.0 ml
    5. Propylene Glycol 40.0 ml
    6. Alcohol 40.0 ml
    7. Purified Water, sufficient quantity to make 100.0 ml
  • Dissolve 1 in 4 and 5 which have been heated to 50°C. Add 2 and 3 to the alcohol and mix with 1 at 25°C. Add sufficient water to make the product measure 100 ml.
    • EXAMPLE 17
  • Colloidal Magnesium Aluminum Premix (5% fully hydrated magma)
    Ingredient Quantity
    1. Colloidal magnesium aluminum silicate 5.0% w/v
    2. Methyl paraben 0.12% w/v
    3. Propyl paraben 0.03% w/v
    4. Water, qs 100%
    • A. The parabens are dissolved in approximately 60% of the purified water at 90°C.
    • B. The colloidal magnesium aluminum silicate is slowly added to step A and maintained at 90°C for one hour with gentle agitation.
    • C. The premix is cooled to 40°C and passed through a colloid mill or homogenizer (2500 psi) rinsing through with fresh purified water.
    • D. The premix is brought to final volume with purified water and agitation.
    • E. The premix may be stored in suitable containers at room temperature for several months or more.
    EXAMPLE 18
  • Oral Suspension
    Ingredient Quantity
    1. Withania Somnifera Extract 0.3% w/v
    2. Colloidal magnesium aluminum silicate premix (5% formula 21) 20.0% w/v
    3. Poloxamer 331 0.05% w/v
    4. Glycerin 10.0% w/v
    5. Potassium sorbate 0.2% w/v
    6. Sodium benzoate 0.1 % w/v
    7. Colorant qs
    8. Flavor qs
    9. Liquid sugar (sp. Gr = 1.33) 65.0%
    10. Citric acid or sodium hydroxide to pH 5.5 qs
    11. Purified water, qs 100%
    1. Dissolve potassium sorbate, sodium benzoate, colorant in aqueous glycerin.
    2. Add liquid sugar colloidal magnesium aluminum silicate premix and half the poloxamer 331 and sulfa drug in step 2 with agitation.
    3. Disperse the rest of the poloxamer 331 and sulfa drug in step 2 with agitation.
    4. 4. Add flavor to step 3 and pass the suspension through a colloid mill or homogenizer rinsing through with purified water.
    5. Adjust pH to 5.5 with either citric acid or sodium hydroxide solution.
    6. Add purified water to final volume.
    • EXAMPLE 19 Improved Bioactivity in the Presence of Oligosaccharides Test Protocol :
  • Ehrlich Ascites tumor (s-180, 1 x 106 cells, all viable), suspended in phosphate buffer saline (PBS, 0.5 ml), were inoculated (i.p.) to a group of adult mice. After 2 h, withaferin-A (WA) (2.5 mg/100 g b.w.) and withanolide + oligosaccharides (1:10 w/w) were administered to Group-2 and Group-3 animals (Table 3). The control (Group 1) received only the vehicle (PBS) following the tumor inoculation. On day-10, the tumor cells were removed from the peritoneal cavity and the viable and dead cells were enumerated microscopically using the dye-exclusion method.
    • Effect of Withaferin A on the Viabilitv and Growth of S-180 Tumor Cells
  • TABLE 3
    Groupa/Treatment Tumor cells x 107 Viable Cells ± SD Dead Cells
    1. PBS (Control) 12.14 ± 3.04 0.80 ± 0.04
    2. Withaferin A (WA) 8.49 ± 2.15 5.17 ± 1.97
    3. VVithaferin-A + Oligosaccharides (1:10) 3.03 ± 0.92 0.72 ± 0.05
    a 12-week-old Swiss mice; n = 10 in each group; WA, withaferin-A: and withaferin-A + oligosaccharides of W. somnifera were administered.
  • The results in Table 3 suggest that the anti-tumor activity of Group 3 withaferin A + oligosaccharides (1:10), unlike that of WA, was not due to cytotoxicity common to anti-tumor drugs, but due to immunomodulation (Ghosal et al., Phytother. Res., 5, 201-206, 1989). This postulate is supported by the decreased tumor growth and only a marginal increase in the number of dead cells compared to the control group. By contrast, the WA group elicited less effective tumor control compared to Group-3 and an appreciable number of dead cells.
  • Thus, the oligosaccharide carrier Group 3 in the extract composition of the invention can significantly modify the nature and extent of the bioactivity of withaferin-A (WA).
  • In summary, the Withania Somnifera extract composition of the invention includes an advantageous combination of components in defined amounts and proportions for optimum biological activity. Particularly, the sitoindoside constituent therein produces an immunostimulation response as reflected by activation of peritoneal macrophages, phagocytosis, and increased activity of lysomal enzymes secreted by activated macrophages. The substantial absence of withasteroid aglycones therein precludes the adverse cytotoxic effect observed with other related compositions in the art. The defined amount and kind of oligosaccharides in the extract composition of the invention also plays a very important role in the bioavailability of the Withania Sominifera active principles. Specifically, the combination of such oligosaccharides without Withanolide aglycones elicits a desirable immunostimulatory effect for the user.

Claims (9)

  1. A Withania Somnifera extract composition obtainable by extraction with a 1:1 aqueous-alcoholic solvent, said extract being in the form of a stable, herbaceous powder, which produces a cognition effect and learning facility for the user, when taken in a dosage of 200-800 mg/day, which comprises, by weight, (a) at least 3% of withanolide glycosides including sitoindosides, (b) at least 3% of oligosaccharides, and (c) less than 0.5% of free withaferin A (aglycone), wherein the weight ratio of (a):(c) is 75-95:25-5, and the weight ratio of (a),(b) is 40-60:60-40.
  2. A composition according to claim 1 wherein the oligosaccharides have a molecular weight of less than 2,000.
  3. A composition according to claim 2 including ash wherein said extract composition is at least 90% water soluble, the ash content is a maximum of 8%, and its moisture content is a maximum of 5% (w/w).
  4. A composition according to claim 1 wherein (a) is 3-8%, and (b) is 3-8%.
  5. A process of making the extract composition of claim 1 which comprises (a) providing root stock of a Withania Somnifera plant which is about 1-2 years old, (b) extracting said root stock immediately with an aqueous-alcoholic solvent, (c) concentrating the extract under vacuum, (d) treating the residue with an apolar organic solvent to remove free withanolide A aglycones therefrom, (e) vacuum drying the insoluble residue of such treatment below about 60°C to provide a dry solid, and (f) pulverizing the solid under controlled temperature and humidity conditions, to obtain the desired powder product.
  6. A process according to claim 5 wherein said aqueous-alcoholic solvent is water-methanol or water-ethanol, and said organic solvent is chloroform or ethyl acetate.
  7. A pharmaceutical or nutritional use product which includes the extract composition of any of claims 1 to 4 in a dosage form suitable for oral administration at a dosage level is 200 to 800 mg/day, optionally including, excipients suitable for such oral administration, wherein the dosage form is a tablet, a capsule or an elixir or suspension.
  8. A product according to claim 7 which is a vitamin tablet.
  9. Use of a composition as claimed in any of claims 1 to 4 in a process for making a pharmaceutical product for immuno-stimulation.
EP00982710A 1999-07-13 2000-07-11 Withania somnifera composition Expired - Lifetime EP1210095B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US351890 1999-07-13
US09/351,890 US6153198A (en) 1999-07-13 1999-07-13 Withania somnifera composition
PCT/US2000/018851 WO2001003715A1 (en) 1999-07-13 2000-07-11 Withania somnifera composition

Publications (3)

Publication Number Publication Date
EP1210095A1 EP1210095A1 (en) 2002-06-05
EP1210095A4 EP1210095A4 (en) 2003-05-02
EP1210095B1 true EP1210095B1 (en) 2006-09-06

Family

ID=23382856

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00982710A Expired - Lifetime EP1210095B1 (en) 1999-07-13 2000-07-11 Withania somnifera composition

Country Status (8)

Country Link
US (1) US6153198A (en)
EP (1) EP1210095B1 (en)
AT (1) ATE338562T1 (en)
AU (1) AU2318601A (en)
CA (1) CA2374929A1 (en)
DE (1) DE60030568T2 (en)
ES (1) ES2272343T3 (en)
WO (1) WO2001003715A1 (en)

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040033273A1 (en) * 2001-02-14 2004-02-19 Ayurcore, Inc. Withasol and methods of use
WO2003068251A1 (en) * 2002-02-14 2003-08-21 Dalmia Centre For Research And Development Herbal formulation for treating attention defienciency disorder (add/adht) and process for preparation
WO2003079812A1 (en) * 2002-03-25 2003-10-02 Council Of Scientific And Industrial Research Herbal nutraceuticals and a process for preparing the same
NL1021288C2 (en) * 2002-08-16 2004-02-17 Exenta B V Herbal extract, as well as the application of such an herbal extract.
US6713092B1 (en) * 2002-12-03 2004-03-30 Natreon Inc. Withania Somnifera composition, method for obtaining same and pharmaceutical, nutritional and personal care formulations thereof
US7282593B2 (en) * 2003-09-11 2007-10-16 Board Of Trustees Of Michigan State University Withanamide and withanolide compositions and method of use thereof
WO2005077392A1 (en) * 2004-01-19 2005-08-25 Ranbaxy Laboratories Limited Herbal formulation comprising extracts of withania, tinospora and picrorhiza as a pediatric tonic
WO2005092356A1 (en) * 2004-03-25 2005-10-06 Palaniappan Meenakshisundaram A novel herbal composition for treating hiv/aids and fungal infections secondary to hiv
US20050266100A1 (en) * 2004-03-30 2005-12-01 Council Of Scientific And Industrial Research Rafi Marg Process isolation of withaferin-A from plant materials and products therefrom
US7108870B2 (en) * 2004-04-13 2006-09-19 Council Of Scientific & Industrial Research Process for isolation of withaferin-A from plant materials and products therefrom
WO2006067796A1 (en) * 2004-12-24 2006-06-29 Council Of Scientific And Industrial Research Herbal formulation as memory enhacher in alzheimer condition
US7250181B2 (en) * 2005-01-19 2007-07-31 Natreon, Inc. Polyherbal compositions and methods for treating viral infections
US20070036873A1 (en) * 2005-07-27 2007-02-15 Shibnath Ghosal Method of treatment or management of stress
US20090311367A1 (en) * 2008-06-17 2009-12-17 Perry Stephen C Dietary Supplement
WO2010150275A2 (en) * 2009-05-22 2010-12-29 Bio-Ved Pharmaceuticals, Pvt. Ltd. Method of extraction from withania somnifera and one or more fractions containing pharmacologically active ingredients obtained therefrom
WO2011056549A1 (en) * 2009-10-27 2011-05-12 Access Business Group International Llc Molecular targets and dietary modulators of exercise-induced muscle damage
WO2012020422A1 (en) * 2010-08-09 2012-02-16 Interdisciplinary School Of Indian System Of Medicine A novel herbal formulation advocated for the prevention and management of coronary heart disease
WO2012160569A1 (en) 2011-05-23 2012-11-29 Gufic Biosciences Limited "process for extraction of ashwagandha (withania somnifera) roots"
US8597697B2 (en) 2011-06-24 2013-12-03 Nutragenesis, Llc Composition of beta-glucan and ashwagandha
US9084800B2 (en) * 2011-11-07 2015-07-21 Natreon, Inc. Indolealkylamino-withasteroid conjugates and method of use
US8609162B2 (en) * 2012-10-04 2013-12-17 Invivo Beverages Llc Integrated neuromodulation system for mood enhancement of a living human subject
AU2015326378B2 (en) * 2014-09-29 2021-01-21 Council Of Scientific & Industrial Research A formulation useful for delivery of neuro protecting agent
CA3002624A1 (en) 2015-10-22 2017-04-27 Benny Antony A process to enhance the bioactivity of ashwagandha extracts
US9987323B2 (en) 2015-10-22 2018-06-05 Benny Antony Process to enhance the bioactivity of Ashwagandha extracts
EP3585400A4 (en) * 2017-02-27 2020-12-30 University of Pittsburgh - Of the Commonwealth System of Higher Education Anti-psychotic composition and treatment methods
DE102021200826A1 (en) 2021-01-29 2022-08-04 Eva Süßmann composition

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5494668A (en) * 1994-07-11 1996-02-27 Patwardhan; Bhushan Method of treating musculoskeletal disease and a novel composition therefor
US5888514A (en) * 1997-05-23 1999-03-30 Weisman; Bernard Natural composition for treating bone or joint inflammation

Also Published As

Publication number Publication date
EP1210095A1 (en) 2002-06-05
ATE338562T1 (en) 2006-09-15
EP1210095A4 (en) 2003-05-02
DE60030568D1 (en) 2006-10-19
WO2001003715A1 (en) 2001-01-18
ES2272343T3 (en) 2007-05-01
DE60030568T2 (en) 2007-09-13
AU2318601A (en) 2001-01-30
CA2374929A1 (en) 2001-01-18
US6153198A (en) 2000-11-28

Similar Documents

Publication Publication Date Title
EP1210095B1 (en) Withania somnifera composition
US7318938B2 (en) Withania somnifera composition, method for obtaining same and pharmaceutical, nutritional and personal care formulations thereof
EP0282002B1 (en) Compositions of oats and nettle extracts to be used as a food additive or pharmaceutical preparation in human health care
DE112009005072T5 (en) A process for producing an extract of Gynostaema pentaphyllum having elevated levels of damulin A and damulin B and pharmaceutical compositions thereof for the treatment of metabolic syndrome
CN101156883A (en) Stevia rebaudian valid target as well as its activity and application
CN106963853A (en) A kind of Rhizoma Belamcandae total-flavonoid aglycone extract and its production and use
JPH045652B2 (en)
CN101496837A (en) &#39;Relingqing&#39; chewable tablet and preparation method thereof
CN1565467B (en) Use of cornel and its extract in preparation alpha-glucosidase inhibitor medicine
CN101948453B (en) Novel NEO-clerodane typed diterpene compound and application thereof
JP2000344675A (en) Prophylactic agent for alcohol damage, and food containing the same
KR102604237B1 (en) Composition for enhancing the bioavailability of fat-soluble vitamins
CN112294883B (en) Stomach-strengthening tablet medicinal preparation and preparation method thereof
CN110117332A (en) A kind of isolated sposknikovan and application thereof
CN101108224A (en) Plants natural base extractive and formulated product and use thereof
KR100477957B1 (en) Novel pyrrole derivatives isolated from lycium chinense mill having hepatoprotective activity and composition containing same
CN1923191B (en) Use of flavanone kind composition in preparation of medicine for curing cardio vascular diseases
KR100485936B1 (en) Anticarcinogenic constituents of ginsenoside Rh2 and Rg3
CN110143989A (en) A kind of novel Ellagitannins class alpha-glucosidase restrainer and preparation method thereof
KR20030084036A (en) Boi tea compositions having anti-obesity and anti-hypercholesterolemia
KR102134389B1 (en) New benzonitrile glycoside compounds and compositions for preventing or treating cancer comprising them
KR101987971B1 (en) Composition for Preventing or Treating Arthritis, Comprising Aromatic Ketone Compound, Melicope pteleifolia Extract, or Fraction Thereof Containing the Same
KR100504021B1 (en) Solid dispersions of insoluble drug and oral antifungal compositions containing them
KR101995244B1 (en) New egcg glycosides and method for preparing the same
KR20230153536A (en) Anti-inflammatory composition comprising biorenovation extract of Lycium chinense sprout as effective component

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020116

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

A4 Supplementary search report drawn up and despatched

Effective date: 20030317

17Q First examination report despatched

Effective date: 20030730

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 36/81 20060101AFI20060209BHEP

Ipc: A61K 125/00 20060101ALI20060209BHEP

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20060906

Ref country code: LI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

Ref country code: CH

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 60030568

Country of ref document: DE

Date of ref document: 20061019

Kind code of ref document: P

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061206

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061206

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070219

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
ET Fr: translation filed
REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2272343

Country of ref document: ES

Kind code of ref document: T3

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20070607

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070731

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20061207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070711

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20070711

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20060906

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20150729

Year of fee payment: 16

Ref country code: GB

Payment date: 20150727

Year of fee payment: 16

Ref country code: ES

Payment date: 20150727

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20150717

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20150727

Year of fee payment: 16

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 60030568

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20160711

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170201

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160801

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20170331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160711

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160711

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160712

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20181126